An analysis of the functional role of a diacidic motif (Asp 236-Asp237)) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Young II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by ∼40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by ∼30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Young II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Young II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Oct 12 2001|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology